Impaired hemodynamics of the patella in patients with patellofemoral pain: A case-control study.

Purpose: According to the homeostasis model, patellofemoral pain (PFP) arises as a consequence of disturbed homeostasis of anterior structures of the knee due to vascular insufficiency. Near-infrared spectroscopy (NIRS) allows to measure changes of concentrations (µmol/cm2) of (de)-oxygenated hemoglobine (HHb and O2Hb). The aim was to study differences in patellar hemodynamics between patients and healthy controls. Methods: Hemodynamics of patients (n = 30 [female = 20, age = 21.5, BMI = 22.9]) and controls (n = 30 (female = 18, age = 21.4, BMI = 22.4]) were evaluated for two activities ('Prolonged Sitting' and 'Stair Descent'). Blinding for health status was implemented. Results: During 'Prolonged Sitting', PFP patients exhibited smaller decreases in mean changes for HHb (PFP [M = -1.5 to -1.9], healthy controls [M = -2.0 to -2.3]) and O2Hb (PFP [M = -2.0 to -3.2], healthy controls [M = -3.4 to -4.1]). However, these differences were statistically non-significant (p = 0.14-0.82 and p = 0.056-0.18, respectively). Conversely, for 'Stair Descent', PFP patients showed statistically significant smaller decreases in mean changes for HHb (PFP [M = -1.9, SD = 1.8], healthy controls [M = -2.5, SD = 1.7], p = 0.043) and O2Hb (PFP [M = -3.2, SD = 3.2], healthy controls [M = -4.9, SD = 2.7], p = 0.004). Conclusions: The differences suggest potential impairment in patellar hemodynamics in PFP patients, providing support for the homeostasis model. Evidence-based treatment strategies targeting patellar hemodynamics should be further refined and subjected to evaluation in clinical trials. Level of Evidence: Level III.

Patients with patellofemoral pain have lower soft tissue flexibility of the kinetic chain compared to healthy controls: A case-control study.

INTRODUCTION: Patellofemoral pain (PFP) is a common musculoskeletal condition. Lower limb range of motion (LLROM) evaluates soft tissue flexibility over multiple joints as part of the kinetic chain. The aims were to study: 1) the reliability of a new LLROM test; 2) differences in LLROM between PFP patients and controls; and 3) the relationship between LLROM and pain-free knee function. METHODS: Patients with PFP and matched controls were recruited from a university campus and private physiotherapy clinics, while observers were blinded for health status. Testing LLROM for maximal knee flexion and hip adduction and the sum of these (total ROM) were performed. Measures of reliability (ICC2,1) were established. Univariate linear regression between LLROM and health status and multivariate analysis between LLROM and knee function were performed. RESULTS: Patients (n = 32 (7 male/25 female, age = 22, BMI = 22.7)) and controls (n = 32 (7 male/25 female, age = 20, BMI = 22.3)) were included. The ICC's for intra- and interobserver reliability ranged from 0.83 (95%CI 0.30-0.93) to 0.89 (0.72-0.95). Symptomatic legs had 7°(3-11, p = 0.014) lower knee flexion, 6°(4-8, p ≤ 0.001) lower hip adduction and 13°(8-17, p ≤ 0.001) lower total ROM than non-symptomatic legs. Multivariate analysis revealed an association between total ROM and pain-free knee function (R2 = 0.438, F = 6.544, p ≤ 0.001). CONCLUSIONS: The new LLROM test was found to be reliable. Patients with PFP had lower LLROM, which was associated with impaired pain-free knee function. Whether improving soft tissue flexibility results in enhanced pain-free knee function should be the subject of future research.

Near-Infrared Spectroscopy measurements are reliable for studying patellar bone hemodynamics and affected by venous occlusion, but not by skin compression.

PURPOSE: According to the homeostasis model, patellofemoral pain (PFP) results from disturbed homeostasis due to vascular insufficiency in the anterior knee. Near-Infrared Spectroscopy (NIRS) measures relative changes in concentrations (in µmol/cm2) of (de-)oxygenated hemoglobine (HHb and O2Hb). The aims were to: 1) investigate the characteristics of the NIRS signal derived from the patella during experiments affecting hemodynamics in healthy controls, and 2) determine the test-retest reliability of NIRS in positions clinically relevant for PFP patients. METHODS: Two experiments were conducted on 10 healthy controls and analysed using Student's t-test. Reliability (ICC2,1) was evaluated for two activities ('Prolonged Sitting' and 'Stair Descent') in five PFP patients and 15 healthy controls, performed twice within five days. RESULTS: The NIRS signal (HHb and O2Hb) showed a statistically significant increase (p < .001 - .002) on all optodes (30, 35, 40 mm) during 'Venous Occlusion' (M = 1.0 - 2.0), while it showed no statistically significant change (p = .075 - .61) during 'Skin Compression' (M = -0.9 - 0.9) on the 30 and 35 mm optode. Reliability of NIRS (HHb and O2Hb) ranged from moderate to almost perfect (ICC2,1 = .47 - .95) on the 30 mm optode for 'Prolonged Sitting', and from moderate to substantial (ICC2,1 = .50 - .68) on the 35 mm optode for 'Stair Descent'. CONCLUSIONS: Patella NIRS measurements are affected by venous occlusion, but not by skin compression, and are sufficiently reliable as research application to compare real-time patellar bone hemodynamics. These insights may assist to improve effectiveness of evidence-based treatment strategies for PFP. TRIAL REGISTRATION: ISRCTN Trial Registration under number: 90377123.

Subsequent AS01-adjuvanted vaccinations induce similar transcriptional responses in populations with different disease statuses.

Transcriptional responses to adjuvanted vaccines can vary substantially among populations. Interindividual diversity in levels of pathogen exposure, and thus of cell-mediated immunological memory at baseline, may be an important determinant of population differences in vaccine responses. Adjuvant System AS01 is used in licensed or candidate vaccines for several diseases and populations, yet the impact of pre-existing immunity on its adjuvanticity remains to be elucidated. In this exploratory post-hoc analysis of clinical trial samples (clinicalTrials.gov: NCT01424501), we compared gene expression patterns elicited by two immunizations with the candidate tuberculosis (TB) vaccine M72/AS01, between three groups of individuals with different levels of memory responses to TB antigens before vaccination. Analyzed were one group of TB-disease-treated individuals, and two groups of TB-disease-naïve individuals who were (based on purified protein derivative [PPD] skin-test results) stratified into PPD-positive and PPD-negative groups. Although TB-disease-treated individuals displayed slightly stronger transcriptional responses after each vaccine dose, functional gene signatures were overall not distinctly different between groups. Considering the similarities with the signatures found previously for other AS01-adjuvanted vaccines, many features of the response appeared to be adjuvant-driven. Across groups, cell proliferation-related signals at 7 days post-dose 1 were associated with increased anti-M72 antibody response magnitudes. These early signals were stronger in the TB-disease-treated group as compared to both TB-disease-naïve groups. Interindividual homogeneity in gene expression levels was also higher for TB-disease-treated individuals post-dose 1, but increased in all groups post-dose 2 to attain similar levels between the three groups. Altogether, strong cell-mediated memory responses at baseline accelerated and amplified transcriptional responses to a single dose of this AS01-adjuvanted vaccine, resulting in more homogenous gene expression levels among the highly-primed individuals as compared to the disease-naïve individuals. However, after a second vaccination, response heterogeneity decreased and was similar across groups, irrespective of the degree of immune memory acquired at baseline. This information can support the design and analysis of future clinical trials evaluating AS01-adjuvanted vaccines.

Short-term effectiveness of an intervention targeting lower limb range of motion on pain and disability in patellofemoral pain patients: A randomized, non-concurrent multiple-baseline study.

INTRODUCTION: Patellofemoral pain (PFP) is a common and often long-standing musculoskeletal condition. Evidence of the effectiveness of interventions addressing soft tissue flexibility is conflicting and of inconsistent scientific quality. However, reduced soft tissue flexibility can negatively affect patellofemoral joint kinematics. Lower limb range of motion (LLROM) reflects soft tissue flexibility throughout the kinetic chain. The aim was to evaluate the short-term effectiveness of an intervention targeting LLROM on pain and disability in patients with PFP. METHODS: A randomized, non-concurrent, multiple-baseline single-case design with a two-week intervention phase and baseline and postintervention phase with varying length was conducted. Eight participants (5 females, 3 males) of age 19(±1.6) years, weekly sports participation 12(±3.1) hours and 17(±14) months symptom duration were included. The Anterior Knee Pain Scale - Dutch Version (AKPS-DV) and the Patient Specific Complaint Scale (PSCS) were administered twice a week. After allocating participants to one of four subgroups of reduced LLROM the intervention was applied. The intervention consisted of soft tissue techniques (mobilization, taping, and stretching). RESULTS: Participant 3 and 6 showed a medium and small but statistically significant positive effect on the AKPS-DV. Participant 2 showed a large and statistically significant positive effect on the PSCS. CONCLUSIONS: This study provides moderate evidence that an intervention targeting LLROM in patients with PFP reduces pain and disability in the short-term. Further research is needed to evaluate the long-term effectiveness and optimize individual treatment outcomes.

Innate Immune Responses to Chimpanzee Adenovirus Vector 155 Vaccination in Mice and Monkeys.

Replication-deficient chimpanzee adenovirus (ChAd) vectors represent an attractive vaccine platform and are thus employed as vaccine candidates against several infectious diseases. Since inducing effective immunity depends on the interplay between innate and adaptive immunity, a deeper understanding of innate immune responses elicited by intramuscularly injected ChAd vectors in tissues can advance the platform's development. Using different candidate vaccines based on the Group C ChAd type 155 (ChAd155) vector, we characterized early immune responses in injected muscles and draining lymph nodes (dLNs) from mice, and complemented these analyses by evaluating cytokine responses and gene expression patterns in peripheral blood from ChAd155-injected macaques. In mice, vector DNA levels gradually decreased post-immunization, but local transgene mRNA expression exhibited two transient peaks [at 6 h and Day (D)5], which were most obvious in dLNs. This dynamic pattern was mirrored by the innate responses in tissues, which developed as early as 1-3 h (cytokines/chemokines) or D1 (immune cells) post-vaccination. They were characterized by a CCL2- and CXCL9/10-dominated chemokine profile, peaking at 6 h (with CXCL10/CCL2 signals also detectable in serum) and D7, and clear immune-cell infiltration peaks at D1/D2 and D6/D7. Experiments with a green fluorescent protein-expressing ChAd155 vector revealed infiltrating hematopoietic cell subsets at the injection site. Cell infiltrates comprised mostly monocytes in muscles, and NK cells, T cells, dendritic cells, monocytes, and B cells in dLNs. Similar bimodal dynamics were observed in whole-blood gene signatures in macaques: most of the 17 enriched immune/innate signaling pathways were significantly upregulated at D1 and D7 and downregulated at D3, and clustering analysis revealed stronger similarities between D1 and D7 signatures versus the D3 signature. Serum cytokine responses (CXCL10, IL1Ra, and low-level IFN-α) in macaques were predominantly observed at D1. Altogether, the early immune responses exhibited bimodal kinetics with transient peaks at D1/D2 and D6/D7, mostly with an IFN-associated signature, and these features were remarkably consistent across most analyzed parameters in murine tissues and macaque blood. These compelling observations reveal a novel aspect of the dynamics of innate immunity induced by ChAd155-vectored vaccines, and contribute to ongoing research to better understand how adenovectors can promote vaccine-induced immunity.

Transcriptional profiles of adjuvanted hepatitis B vaccines display variable interindividual homogeneity but a shared core signature.

The current routine use of adjuvants in human vaccines provides a strong incentive to increase our understanding of how adjuvants differ in their ability to stimulate innate immunity and consequently enhance vaccine immunogenicity. Here, we evaluated gene expression profiles in cells from whole blood elicited in naive subjects receiving the hepatitis B surface antigen formulated with different adjuvants. We identified a core innate gene signature emerging 1 day after the second vaccination and that was shared by the recipients of vaccines formulated with adjuvant systems AS01B, AS01E, or AS03. This core signature associated with the magnitude of the hepatitis B surface-specific antibody response and was characterized by positive regulation of genes associated with interferon-related responses or the innate cell compartment and by negative regulation of natural killer cell-associated genes. Analysis at the individual subject level revealed that the higher immunogenicity of AS01B-adjuvanted vaccine was linked to its ability to induce this signature in most vaccinees even after the first vaccination. Therefore, our data suggest that adjuvanticity is not strictly defined by the nature of the receptors or signaling pathways it activates but by the ability of the adjuvant to consistently induce a core inflammatory signature across individuals.

Modeling adaptive response profiles in a vaccine clinical trial.

BACKGROUND: Vaccine clinical studies typically provide time-resolved data on adaptive response read-outs in response to the administration of that particular vaccine to a cohort of individuals. However, modeling such data is challenged by the properties of these time-resolved profiles such as non-linearity, scarcity of measurement points, scheduling of the vaccine at multiple time points. Linear Mixed Models (LMM) are often used for the analysis of longitudinal data but their use in these time-resolved immunological data is not common yet. Apart from the modeling challenges mentioned earlier, selection of the optimal model by using information-criterion-based measures is far from being straight-forward. The aim of this study is to provide guidelines for the application and selection of LMMs that deal with the challenging characteristics of the typical data sets in the field of vaccine clinical studies. METHODS: We used antibody measurements in response to Hepatitis-B vaccine with five different adjuvant formulations for demonstration purposes. We built piecewise-linear, piecewise-quadratic and cubic models with transformations of the axes with pre-selected or optimized knot locations where time is a numerical variable. We also investigated models where time is categorical and random effects are shared intercepts between different measurement points. We compared all models by using Akaike Information Criterion (AIC), Bayesian Information Criterion (BIC), Deviance Information Criterion (DIC), variations of conditional AIC and by visual inspection of the model fit in the light of prior biological information. RESULTS: There are various ways of dealing with the challenges of the data which have their own advantages and disadvantages. We explain these in detail here. Traditional information-criteria-based measures work well for the coarse selection of the model structure and complexity, however are not efficient at fine tuning of the complexity level of the random effects. CONCLUSIONS: We show that common statistical measures for optimal model complexity are not sufficient. Rather, explicitly accounting for model purpose and biological interpretation is needed to arrive at relevant models. TRIAL REGISTRATION: Clinical trial registration number for this study: NCT00805389, date of registration: December 9, 2008 (pro-active registration).

The Ratiometric Transcript Signature MX2/GPR183 Is Consistently Associated With RTS,S-Mediated Protection Against Controlled Human Malaria Infection.

The RTS,S/AS01 vaccine provides partial protection against Plasmodium falciparum infection but determinants of protection and/or disease are unclear. Previously, anti-circumsporozoite protein (CSP) antibody titers and blood RNA signatures were associated with RTS,S/AS01 efficacy against controlled human malaria infection (CHMI). By analyzing host blood transcriptomes from five RTS,S vaccination CHMI studies, we demonstrate that the transcript ratio MX2/GPR183, measured 1 day after third immunization, discriminates protected from non-protected individuals. This ratiometric signature provides information that is complementary to anti-CSP titer levels for identifying RTS,S/AS01 immunized people who developed protective immunity and suggests a role for interferon and oxysterol signaling in the RTS,S mode of action.

Identification of potential biomarkers of vaccine inflammation in mice.

Systems vaccinology approaches have been used successfully to define early signatures of the vaccine-induced immune response. However, the possibility that transcriptomics can also identify a correlate or surrogate for vaccine inflammation has not been fully explored. We have compared four licensed vaccines with known safety profiles, as well as three agonists of Toll-like receptors (TLRs) with known inflammatory potential, to elucidate the transcriptomic profile of an acceptable response to vaccination versus that of an inflammatory reaction. In mice, we looked at the transcriptomic changes in muscle at the injection site, the lymph node that drained the muscle, and the peripheral blood mononuclear cells (PBMCs)isolated from the circulating blood from 4 hr after injection and over the next week. A detailed examination and comparative analysis of these transcriptomes revealed a set of novel biomarkers that are reflective of inflammation after vaccination. These biomarkers are readily measurable in the peripheral blood, providing useful surrogates of inflammation, and provide a way to select candidates with acceptable safety profiles.

The decline step-down test measuring the maximum pain-free flexion angle: A reliable and valid performance test in patients with patellofemoral pain.

OBJECTIVES: Patients with patellofemoral pain (PFP) experience pain while descending stairs. To date no reliable and valid performance-test exists to assess the maximum pain-free knee flexion angle (MPFFA) as outcome measure during a step-down task. Therefore, the intra- and inter-observer reliability and construct validity of the decline step-down test (DSDT) measuring the MPFFA in patients with PFP were evaluated. DESIGN: Reliability and construct validity. SETTING: Private practices in Nijmegen and Utrecht, the Netherlands. PARTICIPANTS: Patients with PFP. MAIN OUTCOME MEASURES: The reliability was assessed by repeated measurements of the MPFFA during the DSDT. The construct validity was assessed by correlating the measurements on the DSDT with the Anterior Knee Pain Scale Dutch Version (AKPS-DV) based on a pre-set hypothesis. RESULTS: Thirty-two participants (forty-eight knees) were eligible for inclusion. The intraclass correlation coefficient (ICC) for intra-observer reliability was ICC2,1 = 0.83 and ICC2,1 = 0.85 for inter-observer reliability. The 95% limits of agreement (LoA) showed a width of 27.56° for intra-observer reliability and a width of 24.42° for inter-observer reliability. There was an average positive correlation between the DSDT and the total score on the AKPS-DV (rs = 0.31, p = 0.030). CONCLUSION: The DSDT measuring the MPFFA is reliable and valid in patients with PFP.

Characterization of potential biomarkers of reactogenicity of licensed antiviral vaccines: randomized controlled clinical trials conducted by the BIOVACSAFE consortium.

Biomarkers predictive of inflammatory events post-vaccination could accelerate vaccine development. Within the BIOVACSAFE framework, we conducted three identically designed, placebo-controlled inpatient/outpatient clinical studies (NCT01765413/NCT01771354/NCT01771367). Six antiviral vaccination strategies were evaluated to generate training data-sets of pre-/post-vaccination vital signs, blood changes and whole-blood gene transcripts, and to identify putative biomarkers of early inflammation/reactogenicity that could guide the design of subsequent focused confirmatory studies. Healthy adults (N = 123; 20-21/group) received one immunization at Day (D)0. Alum-adjuvanted hepatitis B vaccine elicited vital signs and inflammatory (CRP/innate cells) responses that were similar between primed/naive vaccinees, and low-level gene responses. MF59-adjuvanted trivalent influenza vaccine (ATIV) induced distinct physiological (temperature/heart rate/reactogenicity) response-patterns not seen with non-adjuvanted TIV or with the other vaccines. ATIV also elicited robust early (D1) activation of IFN-related genes (associated with serum IP-10 levels) and innate-cell-related genes, and changes in monocyte/neutrophil/lymphocyte counts, while TIV elicited similar but lower responses. Due to viral replication kinetics, innate gene activation by live yellow-fever or varicella-zoster virus (YFV/VZV) vaccines was more suspended, with early IFN-associated responses in naïve YFV-vaccine recipients but not in primed VZV-vaccine recipients. Inflammatory responses (physiological/serum markers, innate-signaling transcripts) are therefore a function of the vaccine type/composition and presence/absence of immune memory. The data reported here have guided the design of confirmatory Phase IV trials using ATIV to provide tools to identify inflammatory or reactogenicity biomarkers.

Adjuvant-Associated Peripheral Blood mRNA Profiles and Kinetics Induced by the Adjuvanted Recombinant Protein Candidate Tuberculosis Vaccine M72/AS01 in Bacillus Calmette-Guérin-Vaccinated Adults.

Systems biology has the potential to identify gene signatures associated with vaccine immunogenicity and protective efficacy. The main objective of this study was to identify optimal postvaccination time points for evaluating peripheral blood RNA expression profiles in relation to vaccine immunogenicity and potential efficacy in recipients of the candidate tuberculosis vaccine M72/AS01. In this phase II open-label study (NCT01669096; https://clinicaltrials.gov/), healthy Bacillus Calmette-Guérin-primed, HIV-negative adults were administered two doses (30 days apart) of M72/AS01. Twenty subjects completed the study and 18 subjects received two doses. Blood samples were collected pre-dose 1, pre-dose 2, and 1, 7, 10, 14, 17, and 30 days post-dose 2. RNA expression in whole blood (WB) and peripheral blood mononuclear cells (PBMCs) was quantified using microarray technology. Serum interferon-gamma responses and M72-specific CD4+ T cell responses to vaccination, and the observed safety profile were similar to previous trials. Two different approaches were utilized to analyze the RNA expression data. First, a kinetic analysis of RNA expression changes using blood transcription modules revealed early (1 day post-dose 2) activation of several pathways related to innate immune activation, both in WB and PBMC. Second, using a previously identified gene signature as a classifier, optimal postvaccination time points were identified. Since M72/AS01 efficacy remains to be established, a PBMC-derived gene signature associated with the protective efficacy of a similarly adjuvanted candidate malaria vaccine was used as a proxy for this purpose. This approach was based on the assumption that the AS01 adjuvant used in both studies could induce shared innate immune pathways. Subjects were classified as gene signature positive (GS+) or gene signature negative (GS-). Assignments of subjects to GS+ or GS- groups were confirmed by significant differences in RNA expression of the gene signature genes in PBMCs at 14 days post-dose 2 relative to prevaccination and in WB samples at 7, 10, 14, and 17 days post-dose 2 relative to prevaccination. Hence, in comparison with a prevaccination, 7, 10, 14, and 17 days postvaccination appeared to be suitable time points for identifying potentially clinically relevant transcriptome responses to M72/AS01 in WB samples.

Erratum: Author Correction: Cellular and molecular synergy in AS01-adjuvanted vaccines results in an early IFNγ response promoting vaccine immunogenicity.

[This corrects the article DOI: 10.1038/s41541-017-0027-3.].

Different Adjuvants Induce Common Innate Pathways That Are Associated with Enhanced Adaptive Responses against a Model Antigen in Humans.

To elucidate the role of innate responses in vaccine immunogenicity, we compared early responses to hepatitis B virus (HBV) surface antigen (HBsAg) combined with different Adjuvant Systems (AS) in healthy HBV-naïve adults, and included these parameters in multi-parametric models of adaptive responses. A total of 291 participants aged 18-45 years were randomized 1:1:1:1:1 to receive HBsAg with AS01B, AS01E, AS03, AS04, or Alum/Al(OH)3 at days 0 and 30 (ClinicalTrials.gov: NCT00805389). Blood protein, cellular, and mRNA innate responses were assessed at early time-points and up to 7 days after vaccination, and used with reactogenicity symptoms in linear regression analyses evaluating their correlation with HBs-specific CD4+ T-cell and antibody responses at day 44. All AS induced transient innate responses, including interleukin (IL)-6 and C-reactive protein (CRP), mostly peaking at 24 h post-vaccination and subsiding to baseline within 1-3 days. After the second but not the first injection, median interferon (IFN)-γ levels were increased in the AS01B group, and IFN-γ-inducible protein-10 levels and IFN-inducible genes upregulated in the AS01 and AS03 groups. No distinct marker or signature was specific to one particular AS. Innate profiles were comparable between AS01B, AS01E, and AS03 groups, and between AS04 and Alum groups. AS group rankings within adaptive and innate response levels and reactogenicity prevalence were similar (AS01B ≥ AS01E > AS03 > AS04 > Alum), suggesting an association between magnitudes of inflammatory and vaccine responses. Modeling revealed associations between adaptive responses and specific traits of the innate response post-dose 2 (activation of the IFN-signaling pathway, CRP and IL-6 responses). In conclusion, the ability of AS01 and AS03 to enhance adaptive responses to co-administered HBsAg is likely linked to their capacity to activate innate immunity, particularly the IFN-signaling pathway.

Predicting RTS,S Vaccine-Mediated Protection from Transcriptomes in a Malaria-Challenge Clinical Trial.

The RTS,S candidate malaria vaccine can protect against controlled human malaria infection (CHMI), but how protection is achieved remains unclear. Here, we have analyzed longitudinal peripheral blood transcriptome and immunogenicity data from a clinical efficacy trial in which healthy adults received three RTS,S doses 4 weeks apart followed by CHMI 2 weeks later. Multiway partial least squares discriminant analysis (N-PLS-DA) of transcriptome data identified 110 genes that could be used in predictive models of protection. Among the 110 genes, 42 had known immune-related functions, including 29 that were related to the NF-κB-signaling pathway and 14 to the IFN-γ-signaling pathway. Post-dose 3 serum IFN-γ concentrations were also correlated with protection; and N-PLS-DA of IFN-γ-signaling pathway transcriptome data selected almost all (44/45) of the representative genes for predictive models of protection. Hence, the identification of the NF-κB and IFN-γ pathways provides further insight into how vaccine-mediated protection may be achieved.

Axonal transport deficits in multiple sclerosis: spiraling into the abyss.

The transport of mitochondria and other cellular components along the axonal microtubule cytoskeleton plays an essential role in neuronal survival. Defects in this system have been linked to a large number of neurological disorders. In multiple sclerosis (MS) and associated models such as experimental autoimmune encephalomyelitis (EAE), alterations in axonal transport have been shown to exist before neurodegeneration occurs. Genome-wide association (GWA) studies have linked several motor proteins to MS susceptibility, while neuropathological studies have shown accumulations of proteins and organelles suggestive for transport deficits. A reduced effectiveness of axonal transport can lead to neurodegeneration through inhibition of mitochondrial motility, disruption of axoglial interaction or prevention of remyelination. In MS, demyelination leads to dysregulation of axonal transport, aggravated by the effects of TNF-alpha, nitric oxide and glutamate on the cytoskeleton. The combined effect of all these pathways is a vicious cycle in which a defective axonal transport system leads to an increase in ATP consumption through loss of membrane organization and a reduction in available ATP through inhibition of mitochondrial transport, resulting in even further inhibition of transport. The persistent activity of this positive feedback loop contributes to neurodegeneration in MS.

Systems analysis of protective immune responses to RTS,S malaria vaccination in humans.

RTS,S is an advanced malaria vaccine candidate and confers significant protection against Plasmodium falciparum infection in humans. Little is known about the molecular mechanisms driving vaccine immunity. Here, we applied a systems biology approach to study immune responses in subjects receiving three consecutive immunizations with RTS,S (RRR), or in those receiving two immunizations of RTS,S/AS01 following a primary immunization with adenovirus 35 (Ad35) (ARR) vector expressing circumsporozoite protein. Subsequent controlled human malaria challenge (CHMI) of the vaccinees with Plasmodium-infected mosquitoes, 3 wk after the final immunization, resulted in ∼50% protection in both groups of vaccinees. Circumsporozoite protein (CSP)-specific antibody titers, prechallenge, were associated with protection in the RRR group. In contrast, ARR-induced lower antibody responses, and protection was associated with polyfunctional CD4+ T-cell responses 2 wk after priming with Ad35. Molecular signatures of B and plasma cells detected in PBMCs were highly correlated with antibody titers prechallenge and protection in the RRR cohort. In contrast, early signatures of innate immunity and dendritic cell activation were highly associated with protection in the ARR cohort. For both vaccine regimens, natural killer (NK) cell signatures negatively correlated with and predicted protection. These results suggest that protective immunity against P. falciparum can be achieved via multiple mechanisms and highlight the utility of systems approaches in defining molecular correlates of protection to vaccination.

Rotarod motor performance and advanced spinal cord lesion image analysis refine assessment of neurodegeneration in experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is a commonly used experimental model for multiple sclerosis (MS). Experience with this model mainly comes from the field of immunology, while data on its use in studying the neurodegenerative aspects of MS is scarce. NEW METHOD: The aim of this study is to improve and refine methods to assess neurodegeneration and function in EAE. Using the rotarod, a tool used in neuroscience to monitor motor performance, we evaluated the correlation between motor performance, disease severity as measured using a clinical scale and area covered by inflammatory lesions. RESULTS: The included parameters are highly correlated in a non-linear manner, with motor performance rapidly decreasing in the intermediate values of the clinical scale. The relation between motor performance and histopathological damage is exclusively determined by lesions in the ventral and lateral columns, based on a new method of analysis of the entire spinal cord. Using a set of definitions for distinct disease milestones, we quantified disease duration as well as severity. COMPARISON WITH EXISTING METHODS: The rotarod measures motor performance in a more objective and quantitative manner compared to using a clinical score. The outcome shows a strong correlation to the surface area of inflammatory lesions in the motor systems of the spinal cord. CONCLUSIONS: These results provide an improved workflow for interpreting the outcome of EAE from a neurological point of view, with the eventual goal of dissecting neurodegeneration and evaluating neuroprotective drugs in EAE for application in MS.

Resolving bundled microtubules using anti-tubulin nanobodies.

Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50-70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.